Mahoney, Douglas W.

Abstract Introduction: Laparoscopy with peritoneal lavage enhances staging accuracy in patients with pancreatic ductal adenocarcinoma (PDAC). Occult intra-abdominal metastases are identified by staging laparoscopy in approximately 20% of patients with radiologically localized PDAC. Peritoneal fluid cytology has limited sensitivity in detecting occult intra-abdominal metastasis. Methylated DNA markers (MDMs) can accurately detect PDAC from tumor tissue, blood, and pancreatic secretions. This study aims to assess the feasibility of assaying MDMs in peritoneal lavage fluid obtained at the time of staging laparoscopy for patients with PDAC. Methods: Peritoneal lavage fluid (25-60 mL) obtained from patients with PDAC at the time of staging laparoscopy was centrifuged for 10 minutes at 1200 × g. The pellet was resuspended and aliquoted into 200µL tubes and frozen at -80°C. DNA extracted from one aliquot was assayed for mutant KRAS (G12A and G13D) and then bisulfite converted to measure 13 MDMs (GRIN2D, CD1D, ZNF781, FER1L4, RYR2, CLEC11A, AK055957, LRRC4, MAX.CHR5.4295, HOXA1, PRKCB, SHISA9, NTRK3) and a normalizing reference gene (B3GALT6) using quantitative methylation specific PCR. The association of KRAS and the average signal from the 13 MDMs (MDMscore) with intra-abdominal metastasis (Y/N) was assessed with the area under the receiver operator characteristic curve (AUC). Results: Among 48 PDAC patients, staging laparoscopy revealed intra-abdominal metastasis in 15 (31%). Of the 15 intra-abdominal metastases, 6 were found to have biopsy-confirmed intra-abdominal metastasis and positive peritoneal cytology, 4 were positive on peritoneal cytology alone, and 5 had biopsy-confirmed intra-abdominal metastasis alone. The sensitivity of the MDMscore for intra-abdominal metastasis was 87% (CI: 60-98%) at 90% (CI: 76-98%) specificity whereas the sensitivity for KRAS was 40% (CI: 16-68%) at 85% (CI: 68-95) specificity. The AUC for the MDMscore was 0.98 (CI: 0.94-1.00) compared to 0.62 (CI: 0.48-0.77) for that of KRAS (p<0.0001). Conclusion: MDMs assayed from peritoneal lavage fluid collected during staging laparoscopy is a novel tool for accurate molecular staging of PDAC. These early results warrant further study in a larger cohort to assess impact on peritoneal staging and its clinical utility in the neoadjuvant treatment paradigm of PDAC. Citation Format: Kalika Maheshwari, Calise K. Berger, William R. Taylor, Douglas W. Mahoney, Kelli N. Burger, Adriana M. Delgado, Karen A. Doering, Heather M. Streich, Patrick H. Foote, Sean P. Cleary, Travis E. Grotz, Micheal L. Kendrick, Rory L. Smoot, Patrick P. Starlinger, Cornelius A. Thiels, Mark J. Truty, Susanne G. Warner, John B. Kisiel, Shounak Majumder. Methylated DNA markers for detection of peritoneal metastasis in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3784.

Abstract The multi-target stool DNA (mt-sDNA) test screens for colorectal cancer by analyzing DNA methylation/mutation and hemoglobin markers to algorithmically derive a qualitative result. A new panel of highly discriminant candidate methylated DNA markers (MDM) was recently developed. Performance of the novel MDM panel, with hemoglobin, was evaluated in a simulated screening population using archived stool samples weighted to early-stage colorectal cancer and prospectively collected advanced precancerous lesions (APL). Marker selection study (MSS) and separate preliminary independent verification studies (VS) were conducted utilizing samples from multi-center, case–control studies. Sample processing included targeted MDM capture, bisulfite conversion, and MDM quantitation. Fecal hemoglobin was quantified using ELISA. Samples were stratified into 75%/25% training-testing sets; model outcomes were cross-validated 1,000 times. All laboratory operators were blinded. The MSS included 232 cases (120 colorectal cancer/112 APLs) and 490 controls. The VS featured 210 cases (112 colorectal cancer/98 APLs) and 567 controls; APLs were 86.7% adenomas and 13.3% sessile serrated lesions (SSL). Average age was 65.5 (cases) and 63.2 (controls) years. Mean sensitivity in the VS from cross-validation was 95.2% for colorectal cancer and 57.2% for APLs, with specificities of 89.8% (no CRC/APLs) and 92.4% (no neoplasia). Subgroup analyses showed colorectal cancer sensitivities of 93.4% (stage I) and 94.2% (stage II). APL sensitivity was 82.9% for high-grade dysplasia, 73.4% for villous lesions, 49.8% for tubular lesions, and 30.2% for SSLs. These data support high sensitivity and specificity for a next-generation mt-sDNA test panel. Further evaluation of assay performance will be characterized in a prospective, multi-center clinical validation study (NCT04144738). Prevention Relevance: This study highlights performance of the next-generation mt-sDNA test, which exhibits high sensitivity and specificity for detecting colorectal cancer and APLs. This noninvasive option has potential to increase screening participation and clinical outcomes. A multi-center, clinical validation trial is underway. See related commentary by Bresalier, p. 93

Background: Radiographic surveillance of colorectal cancer (CRC) after curative-intent therapy is costly and unreliable. Methylated DNA markers (MDMs) detected primary CRC and metastatic recurrence with high sensitivity and specificity in cross-sectional studies. This study evaluated using serial MDMs to detect recurrence and monitor the treatment response to anti-cancer therapies. Methods: A nested case-control study was drawn from a prospective cohort of patients with CRC who completed curative-intent therapy for CRC of all stages. Plasma MDMs were assayed vis target enrichment long-probe quantitative-amplified signal assays, normalized to B3GALT6, and analyzed in combination with serum carcinoembryonic antigen to yield an MDM score. Clinical information, including treatment and radiographic measurements of the tumor burden, were longitudinally collected. Results: Of the 35 patients, 18 had recurrence and 17 had no evidence of disease during the study period. The MDM score was positive in 16 out of 18 patients who recurred and only 2 of the 17 patients without recurrence. The MDM score detected recurrence in 12 patients preceding clinical or radiographic detection of recurrent CRC by a median of 106 days (range 90–232 days). Conclusions: Plasma MDMs can detect recurrent CRC prior to radiographic detection; this tumor-agnostic liquid biopsy approach may assist cancer surveillance and monitoring.

Plasma-based MDMs may offer a cost-effective alternative to imaging for melanoma detection. Early results show 80% sensitivity and 100% specificity against cancer-free controls. Further study needed. #MelanomaResearch #EarlyDetection

Abstract Lynch syndrome (LS) markedly increases risks of colorectal and endometrial cancers. Early detection biomarkers for LS cancers could reduce the needs for invasive screening and surgical prophylaxis. To validate a panel of methylated DNA markers (MDM) previously identified in sporadic colorectal cancer and endometrial cancer for discrimination of these cancers in LS. In a case–control design, previously identified MDMs for the detection of colorectal cancer and endometrial cancer were assayed by qMSP on tissue-extracted DNA. Results were normalized to ACTB values within each sample. Least absolute shrinkage and selection operator models to classify colorectal cancer and endometrial cancer were trained on sporadic cases and controls and then applied to classify colorectal cancer and endometrial cancer, in those with LS, and cross-validated. We identified colorectal cancer cases (23 with LS, 48 sporadic), colorectal controls (32 LS, 48 sporadic), endometrial cancer cases (30 LS, 48 sporadic), and endometrial controls (29 LS, 37 sporadic). A 3-MDM panel (LASS4, LRRC4, and PPP2R5C) classified LS-CRC from LS controls with an AUC of 0.92 (0.84–0.99); results were similar for sporadic colorectal cancer. A 6-MDM panel (SFMBT2, MPZ, CYTH2, DIDO1, chr10.4479, and EMX2OS) discriminated LS-EC from LS controls with an AUC of 0.92 (0.83–1.0); the AUC for sporadic endometrial cancer versus sporadic controls was nominally higher, 0.99 (0.96–1.0). MDMs previously identified in sporadic endometrial cancer and colorectal cancer discriminate between endometrial cancer and benign endometrium and colorectal cancer and benign colorectum in LS. This supports the inclusion of patients with LS within future prospective clinical trials evaluating endometrial cancer and colorectal cancer MDMs and may provide a new avenue for cancer screening or surveillance in this high-risk population. Prevention Relevance: Lynch syndrome (LS) markedly increases risks of colorectal and endometrial cancers. Early detection biomarkers for LS cancers could reduce the needs for invasive screening and surgery. Methylated DNA markers previously identified in sporadic endometrial cancer and colorectal cancer discriminate between benign and cancer tissue in LS.

Abstract Background: Reliable non-invasive biomarkers for human papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma (OPX) are lacking. We performed a discovery and validation study of methylated DNA markers (MDMs) for detection of OPX. In addition, we sought to evaluate the MDM profiles in saliva gargle samples from normal controls (nSAL). Methods: Formalin fixed paraffin embedded OPX and gender-balanced normal oropharyngeal tissue (NOP) were pathologically reviewed and punched for DNA extraction and sequenced using reduced representation bisulfite sequencing (RRBS). A tiling algorithm was used to define candidate MDMs. Highest ranked (e.g., area under the receiver operating characteristic curve (AUC), fold-change, p-value) candidate MDMs were validated by qMSP in independent OPX and NOP samples. nSAL DNA was used to assess MDM background signal. Discrimination between OPX tissue, NOP, and nSAL was assessed using AUC with corresponding 95% confidence intervals. HPV was confirmed with p16 staining or DNA in situ hybridization. Results: RRBS discovery assessed 18 OPX, 18 NOP, and 18 normal WBC samples. 234 MDM candidates from approximately 3 × 106 CpGs at >10X coverage were identified. 36 MDMs were selected for validation in independent patient samples which included 32 OPX, 36 NOP, and 35 nSAL specimens. OPX patients were older (median 51; IQR 45-54 years) than NOP patients (median 29; IQR 24-42). 69 and 61% of OPX and NOP patients were female, respectively. The majority of OPX patients were Stage I (n=27, 84%). We observed excellent discrimination between OPX and NOP in the validation set; the median AUC across 36 MDMs was 0.90 (IQR 0.87-0.95). The 10 highest performing markers (PARP15, EPDR1, SHROOM1, EMBP1.rs, IFFO1, MAX.chr19.118, ZNF763, ITGB4, FAM19A2, and MT1IP) exhibited AUCs ranging from 0.95-0.99. Strong discrimination was also observed between OPX and nSAL (Median AUC=0.97; IQR=0.93-0.99). All 10 of the highest-performing markers in tissue exhibited AUCs of at least 0.97 for discriminating OPX versus nSAL. Median fold change between NOP and nSAL across all MDMs was 1.9 (IQR 1.4-3.8), demonstrating low background signal in nSAL. Conclusions: We found strong discrimination in a panel of 36 MDMs between OPX case and control tissue as well as between OPX case tissue and normal saliva. The low background signal observed in normal saliva makes this media a strong candidate for non-invasive detection of OPX. Further studies are in development to assess the utility of measuring these MDMs in both case and control saliva and blood for detecting OPX non-invasively. Citation Format: Benjamin R. Gochanour, David M. Routman, Kathleen R. Bartemes, William R. Taylor, Douglas W. Mahoney, Rondell P. Graham, Xiaoming Cao, Calise K. Berger, Sara S. Then, Karen A. Doering, Kelli N. Burger, Patrick H. Foote, John B. Kisiel, Kathryn M. Van Abel. Discovery and validation of methylated DNA markers of human papillomavirus associated oropharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-039.

Abstract Objective: Upper tract urothelial cancers (UTUC) can be difficult to characterize, and non-invasive surveillance tools are lacking. UTUC are often treated with nephroureterectomy (NU). To address these gaps, we sought to discover and validate methylated DNA markers (MDMs) for detection of UTUC. Methods: Archival formalin fixed paraffin embedded (FFPE) tissue samples from NU were pathologically reviewed and punched prior to DNA extraction. Reduced representation bisulfite sequencing (RRBS) was used to identify candidate MDMs that discriminated between UTUC and age-balanced control urothelial tissues (uninvolved ureter/renal pelvic of renal cell carcinoma at radical nephrectomy). Highest ranked (fold-change and p-value) candidate MDMs were biologically validated by quantitative methylation specific PCR in a 2nd independent set of UTUC and age-balanced controls. DNA from healthy donor urine and buffy coat samples was used to assess MDM background signal. Discrimination between UTUC and controls was assessed as the area under the receiver operator characteristic curve (AUC) with corresponding 95% confidence intervals. Results: RRBS assessed 33 UTUC and 26 ureter/renal pelvis controls. 3.01 x 106 CpGs mapped to the reference genome with at least 10X read depth. From 358 candidates, 20 MDMs were selected for biological validation in independent patient samples which included 20 renal pelvis & 16 ureter UTUC and 17 renal pelvis & 15 ureter control specimens. Of UTUC and control patients, 36% and 65% of were men, respectively. Median AUC for comparison of UTUC vs control tissue was 0.87 (IQR 0.81-0.88); AUCs with corresponding 95% CIs are shown for the 10 most accurate MDMs (Table) across all control types. Conclusion: We report discovery and validation of highly sensitive and specific MDMs with potential for accurate non-invasive screening supported by low background in urine and blood. AUCs (95% CI) for selected methylated DNA markers for UTUC vs control tissue, urine, and blood UTUC vs Marker Control Tissue Control Urine Control Buffy C2orf55 0.92 (0.84-1) 0.93 (0.87-1) 0.96 (0.91-1) ANKRD35 0.91 (0.84-0.99) 0.93 (0.86-1) 0.98 (0.94-1) DLX6 0.91 (0.84-0.98) 0.78 (0.66-0.9) 0.97 (0.93-1) Chr8.9998 0.90 (0.82-0.97) 0.91 (0.84-0.97) 0.98 (0.96-1) SP9 0.88 (0.8-0.97) 0.95 (0.9-1) 0.99 (0.98-1) Chr13.1127 0.88 (0.8-0.97) 0.94 (0.89-1) 0.92 (0.85-1) Septin9.r2 0.88 (0.79-0.97) 0.95 (0.88-1) 0.98 (0.94-1) LBX2 0.88 (0.79-0.97) 0.94 (0.88-1) 0.96 (0.91-1) SIM2.r1 0.88 (0.79-0.97) 0.91 (0.82-1) 0.94 (0.86-1) Chr14.1037 0.88 (0.78-0.97) 0.93 (0.85-1) 0.96 (0.9-1) Citation Format: Ojas Bhagra, William R. Taylor, Benjamin R. Gochanour, Patrick H. Foote, Calise K. Berger, Xiaoming Cao, Douglas W. Mahoney, Sara S. Then, Anna M. Gonser, Karen A. Doering, Kelli N. Burger, John C. Cheville, Matthew T. Gettman, John B. Kisiel. Discovery and validation of methylated DNA markers of upper tract urothelial tract cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3349.

Abstract Objective: Aberrantly methylated DNA may be used to detect minimal residual disease or early recurrence in breast cancer. As a 1st step, we aimed to develop an MDM panel to delineate between healthy women, stage I-III breast cancer in remission with no evidence of disease (NED), and metastatic breast cancer (MBC). Methods: We prospectively enrolled patients into 3 groups: healthy controls (adult females with a normal mammogram in past 12 months with no history of breast cancer), NED [stage I-III breast cancer with no clinical evidence of recurrence 6 months - 3 years post-treatment (adjuvant endocrine therapy allowed)], and MBC (collected either at initial diagnosis of untreated metastatic disease or radiographic progression). DNA was extracted from 6 mL of plasma derived from blood collected in LBgard tubes (Exact Sciences Corp., San Diego CA), and was bisulfite-treated and assayed by target enrichment long-probe quantitative amplified signal (TELQASTM) method for a panel of 15 previously identified MDMs [Table], normalized by the control gene, B3GALT6. Areas under the receiver operating characteristic curve (AUC) were calculated for each MDM to assess discrimination of MBC from other groups. With 60 patients per group, we had greater than 80% power to detect a difference of 0.20 from the null AUC of 0.7. Results: We tested samples from 60 MBC (10 untreated, 50 recurrent), 60 NED, and 60 healthy women. The median age was 63 (IQR, 55-72) and was not different between groups. The NED group was all hormone-receptor (HR) positive, with 47 (78%) being human epidermal growth factor receptor 2 negative (HER2-) and 13 (22%) HER2+; 48 (80%) were stage I and 9 (15%) were N+. The MBC group had 35 (58%) patients with HR+/HER2- tumors, 11 (18%) HR+/HER2+, and 6 (10%) HR-/HER2-, 4 (7%) HR-/HER2+, with 4 (7%) unknown. Of the 41 MBC whose original stage and nodal status were known 9 (22%) were stage I and 22 (54%) were N+. The markers were able to discriminate between MBC and NED patient samples with AUCs from 0.97 to 0.72. AUCs for 10/15 MDMs (ITPRIPL1, EMX1, C17orf64, OSR2, TRIM67, CHST2, IFFO1, TRH, FAM59B, and MPZ) were >0.90. No difference in MDMs was observed between the healthy and NED groups. Conclusions: MDMs, assayed from plasma, appear highly discriminant for metastatic breast cancer cases in comparison to control patients with NED or healthy controls. Further clinical validation of this MDM panel in a larger cohort is warranted. Table 1. Plasma MDM discrimination of metastatic breast cancer (MBC) from healthy controls and previously treated breast cancer patients with no evidence of disease (NED) Citation Format: Karthik V. Giridhar, Fergus J. Couch, Jason P. Sinnwell, Seth W. Slettedahl, William R. Taylor, Douglas W. Mahoney, Patrick H. Foote, Maria C. O’Connell, Mariah J. Robran, Mary E. Devens, Anna M. Gonser, Nicole Larson, Karen A. Doering, Kelli N. Burger, Michael Kaiser, Hatim Allawi, Kathryn Ruddy, Janet Olson, John B. Kisiel. Plasma assay of methylated DNA markers (MDM) detects patients with metastatic breast cancer (MBC) compared to healthy controls and treated breast cancer patients with no evidence of disease [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-06.

Abstract Objectives: We hypothesize that a validated panel of methylated DNA markers (MDMs) for HPV(+)CSCC will be similar to but distinct from a cohort of HPV(+)OPSCC. Methods: Patients were excluded for recurrent tumor, prior pelvic or head or neck cancer/neoplasia, chemotherapy < 1 year, prior therapeutic radiation to the target region, prior transplant, insufficient target tissue (<5mm), or if <18 yrs. MDMs identified from sequenced differentially methylated regions were selected from a panel validated for HPV(+)CSCC and evaluated on DNA from independent FFPE HPV(+)OPSCC, HPV(+)CSCC, normal oropharynx tonsil, and normal cervix tissue using methylation-specific PCR. White blood cells (WBCs) were used as a background control. Results: 34 HPV(+)OPSCC, 36 HPV(+)CSCC, 26 normal tonsil tissue, and 24 normal cervical tissue patients met criteria. The HPV(+)OPSCC cohort was older (57 vs 44 yrs, p=0.027), had more frequent alcohol exposure (85% vs 64%, p=0.02), but similar ACE-27 comorbidity scores (p=0.078) and tobacco use (p=0.066). Tumor stages were stage I (47% and 83%), stage II (44% and 11%) and stage III (9% and 6%) for HPV(+)OPSCC and HPV(+)CSCC, respectively. Twenty-one MDMs were evaluated and areas under the receiver operator characteristic curve (AUC) are reported (Table 1). 95% confidence intervals excluded a non-diagnostic null hypothesis AUC of 0.5. 5/21 (24%) achieved an AUC ≥ 0.90, 15/21 (71%) achieved an AUC ≥ 0.8, and 19/21 (90%) exhibited better than chance classifications relative to control tonsil tissue (all p<0.001). Conclusion: MDMs have the potential to discriminate both HPV(+)OPSCC and HPV(+)CSCC from their normal tissue controls. 90% of the MDMs discovered for HPV(+)CSCC had better than chance discrimination in HPV(+)OPSCC suggesting a distinct signature between the two subtypes. Future discovery efforts specific to HPV(+)OPSCC is warranted. Methylated DNA Markers in HPV(+)OPSCC and HPV(+)CSCC CSCC AUC (95% CI) OPSCC AUC (95% CI) AUC CSCC vs AUC OPSCC Pvalue GRIN2D 0.72 (0.59-0.85) 0.91 (0.83-0.99) 0.019 EMX1 0.93 (0.86-0.99) 0.77 (0.65-0.89) 0.0191 VWC2 0.88 (0.79-0.97) 0.57 (0.42-0.72) 0.0001 ZNF610 0.99 (0.98-1) 0.89 (0.8-0.97) 0.0162 ZNF781.F 0.92 (0.86-0.99) 0.79 (0.67-0.91) 0.0585 TBX15 0.99 (0.96-1) 0.82 (0.72-0.93) 0.0025 TSPYL5 0.94 (0.87-1) 0.82 (0.72-0.93) 0.058 LOC645323 0.95 (0.89-1) 0.88 (0.8-0.97) 0.1952 ASCL1 0.98 (0.93-1) 0.86 (0.76-0.95) 0.0187 ABCB1 0.97 (0.93-1) 0.74 (0.61-0.86) 0.0001 ZNF69 0.89 (0.8-0.97) 0.95 (0.9-1) 0.2017 MAX.chr9 0.97 (0.92-1) 0.82 (0.72-0.93) 0.0113 ARHGAP12 0.91 (0.83-0.99) 0.9 (0.81-1) 0.9223 C1orf114 0.94 (0.86-1) 0.88 (0.8-0.97) 0.3366 MAX.chr6 0.99 (0.97-1) 0.86 (0.76-0.96) 0.0099 NEUROG3 0.95 (0.89-1) 0.7 (0.57-0.83) 0.0001 NID2 0.98 (0.95-1) 0.86 (0.77-0.95) 0.0207 TMEM200C 0.95 (0.89-1) 0.54 (0.39-0.69) 0.0001 TTYH1 0.99 (0.98-1) 0.82 (0.71-0.93) 0.0023 ZNF773 0.93 (0.86-1) 0.9 (0.82-0.98) 0.5696 ZNF781.R 1 (1-1) 0.92 (0.86-0.99) 0.0198 Citation Format: Kathryn M. Van Abel, David M. Routman, Daniel J. Ma, Karen A. Doering, Kelli N. Burger, Patrick H. Foote, William R. Taylor, Douglas W. Mahoney, Calise K. Berger, Xiaoming Cao, Sara S. Then, Alyssa M. Larish, Eric J. Moore, Joaquin G. Garcia, Rondell P. Graham, Jamie N. Bakkum-Gamez, John B. Kisiel. Comparison of methylated DNA markers between human papillomavirus mediated carcinoma in oropharynx and cervical cancer: A pilot study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3742.